Recently, this laboratory has demonstrated in vitro that phosphorylation of rabbit skeletal myosin (M) enhances the actin-activated hydrolysis of ATP. These studies will be extended to involve a steady-state kinetic analysis of the affect of phosphorylation on actin-interaction. The cardiac and skeletal system will be compared. The soluble proteolytic subfragments, HMM and S-1 will be prepared in the phosphorylated and nonphosphorylated state. Actin (A) competition experiments between these two species of myosin will determine whether actin-binding in the absence of ATP is altered (the AM complex) as a result of phosphorylation. Lineweaver-Burk plots will compare as a function of the degreee of phosphorylation of myosin, actin-interaction under conditions where A-M.ADP. is the principal actomyosin complex. These two experimental approaches will indicate whether phosphorylation of L2 affects the steady state concentration of AM or A-M.ADP. In addition, we will attempt to render myosin 'soluble' at low ionic strength by cross-linking myosin with one of the di-imido esters. If we are successful in preparing a 'soluble' myosin without altering the hydrolytic site, we will be able to begin a steady state kinetic analysis of the actomyosin system.